Method for obtaining human cdc25 phosphatases and method for identifying human cdc25 phosphatase modulators

ABSTRACT

The invention concerns a method for obtaining human cdc 25B1, cdc25B2, cdcB3 and cdc25C phosphatases. More particularly, the invention concerns a protein for fusion between human cdc25B1, cdc25B2, cdcB3 or cdc25C phosphatase and the maltose binding protein (MBP) of  Escherichia coli , the DNA sequence coding for said fusion protein, a method for preparing said fusion protein and a method for identifying human cdc25B1, cdc25B2, cdcB3 or cdc25C protein modulators.

[0001] The present invention relates to a method for obtaining human Cdc25 phosphatases. It also relates to a method for identifying human Cdc25 phosphatase modulators.

[0002] Entry of the cell into the cell division process is regulated by a group of kinases and phosphatases useful for the synchronisation of the different phases of the cell cycle and allow reorganisation of cell architecture.

[0003] Cycline dependent kinases (CDKs) play a major role in this control and already several inhibitors of this kinase family have been identified. One of these compounds (flavopiridol) is already in clinical Phase II (Senderowicz and Sausville, J. Natl. Cancer Inst. (2000), 92, 376-387).

[0004] These CDKs are activated by dephosphorylation carried out by the Cdc25 phosphatases, on tyrosine and threonine residues. In human cells, the Cdc25 proteins are coded by one family: Cdc25A, Cdc25B and Cdc25C (Cans et al., Medicine Sciences (1998), 3, 269-274).

[0005] A few Cdc25 inhibitors have been identified, but they have only a weak activity (cf. Baratte, B., Meijer, L., Galaktionov, K., and Beach, D., Anticancer Res. (1992), 12, 873-880; Rice, R. L. et al., Biochemistry (1997), 36, 15965-15974; and Ham, S. W., Park, J., Lee, S. J., Kim, W., Kang, K., and Choi, K. H., Bioorg. Med. Chem. Lett. (1998), 8, 2507-2510).

[0006] Cdc25B2 is a protein tyrosine phosphatase similar to Cdc25B phophatase (now called Cdc25B1). It was identified in a DNA bank coding for Burkitt's lymphoma. Cdc25B2 differs from Cdc25B1 by an insertion of 14 amino acids and a deletion of 41 amino acids upstream of the catalytic domain. Cdc25B1 and Cdc25B2 are splicing variants of the same gene. A third variant, Cdc25B3, carrying the two sequences of 14 and 41 amino acids, was identified from the same bank (Baldin et al., Oncogene (1997), 14, 2485-2495). The three variants are detected in the primocultures and the cell lines. Analysis of the variants shows that Cdc25B2 is more weakly expressed than Cdc25B3 in all of the lines tested but that the expression of the two variants increases during the G2 phase and mitosis (Forrest et al., Biochem. Biophys. Res. Commun. (1999), 260, 510-515). Hernandez et al. report that Cdc25A and -B2 but not Cdc25B1, -B3 and -C, are overexpressed in a large number of lymphomas (35% and 39%) (Hernandez et al., Int. J. Cancer (2000), 89(2), 148-52). The normal lymphocytes express the Cdc25B 1 and -B3 messengers and very weakly those of Cdc 25A, -B2 and -C.

[0007] The over-expression of the three Cdc25B variants in yeast shows that Cdc25B2 appears to be more active than B1 or B3 (B2>B3>B1). The alternative splicing of Cdc25B can therefore play a role in the control of cell proliferation.

[0008] Cdc25C phosphatase is moreover itself regulated by phosphorylation on serine-216 by other Cds1 or Chk1 enzymes and binds itself to highly conserved members of the 14-3-3 protein family (Zeng, Y. et al., Nature (1998), 395, 507-510).

[0009] The search for more effective phosphatase inhibitors must overcome the necessity of having a protein which retains its phosphatase activity, in a non-limited quantity so as to allow large scale screening.

[0010] The study of a protein requires large quantities of the latter to satisfy all of the characteristics which can be analysed, in particular in fields such as biophysics (size, sequence, structure etc.), biochemistry (activity, stability, regulation etc.) or pharmacology (activators, inhibitors, etc.).

[0011] The production and the purification of this protein in large quantities comes up against several obstacles which can be:

[0012] absent or too weak an expression of the protein;

[0013] expression of a truncated protein;

[0014] obtaining a protein without biological activity;

[0015] loss of the biological activity of the protein during purification;

[0016] very weak purification yield;

[0017] loss of the biological activity of the protein during storage;

[0018] loss of the production source.

[0019] Only successful completion of each of these stages finally allows the long term production of a biologically-active protein in unlimited quantities. Faced with such requirements, the purification of a number of proteins remains a very delicate even impossible process in some cases.

[0020] However, much effort has been made to develop new production and purification strategies such as:

[0021] large scale cell culture (animal or vegetable);

[0022] large scale microorganism culture (bacteria, yeasts);

[0023] immunoprecipitation from a mixture of proteins using a specific antibody;

[0024] affinity chromatography, using a specific ligand (effectors, repressors, activators);

[0025] bidimensional electrophoresis, as a function of the molecular weight and the isoelectric point of the protein;

[0026] capillary electrophoresis;

[0027] enrichment by differential precipitation with different salts;

[0028] etc.

[0029] Moreover, new systems now try to combine the production and the purification of proteins. These systems allow an often inducible expression of the production of a recombinant protein fused with a protein allowing affinity chromatography (called tag protein). This last part can be eliminated by the addition of a protease which specifically recognises the location of the fusion (Sheibani, N., Prep. Biochem. Biotechnol. (1999), 29, 77-90). The number of systems proposed increases but the success of these different approaches remains very varied depending on the proteins to be purified. The conformation and the solubility of the proteins remain parameters which are impossible to control in these new systems (see Guise, A. D., West, S. M. and Chaudhuri, J. B., Mol. Biotechnol. (1996), 6, 53-64; Kelley, R. F. and Winkler, M. E., Genet. Eng. (N.Y., 1990), 12, 1-19).

[0030] The Cdc25B 1, Cdc25B2, Cdc25B3 or Cdc25C proteins could be fused with systems as varied as:

[0031] 1. The residue of six histidines recognised by six histidine anti-motif antibody (Katsafanas, G. C. and Moss, B., Virology (1999), 258, 469-479);

[0032] 2. The residue of nine amino acids of hemaglutinin from influenza recognised by 3F10 antibodies (Robert, I. and Quirin-Stricker, C., J. Mol. Neurosci. (1998), 11, 243-251);

[0033] 3. The residue of 11 amino acids of the vesicular stomatitis virus recognised by the P5D4 antibody (The Maout, S., et al., Proc. Natl. Acad. Sci. U.S.A. (1997), 94, 13329-13334);

[0034] 4. The residue of 6 amino acids of the capside protein of bovin papilloma virus (AU1) recognised by the anti-AU1 antibody (The Maout, S., et al., Proc. Natl. Acad. Sci. U.S.A. (1997), 94, 13329-13334);

[0035] 5. The residue of 12 amino acids of the heavy chain of the C protein recognised by the HPC4 antibody (Rezaic, A. R., et al., Protein Expr. Purif. (1992), 3, 453-460);

[0036] 6. The C-myc protein recognised by the 9E10 antibody (Bae, S. H., et al., J. Biol. Chem. (1999), 274, 14624-14631);

[0037] 7. The beta galactosidase protein affinity for amino-phenyl-β-D-thiogalactopyranoside (Germino, J. and Bastia, D., Proc. Natl. Acad. Sci. U.S.A. (1984), 81, 4692-4696);

[0038] 8. The gluthation S-transferase protein recognised by the anti-GST antibody (Carr, S., et al., Vaccine (1999), 18, 153-159);

[0039] 9. The biotin-carboxylase carrier has an affinity for avidine (Germino, F. J. and Moskowitz, N. K., Methods Enzymol. (1999), 303, 422-450);

[0040] 10. The intein protein has an affinity for chitin (see Chong, S., et al., Gene (1997), 192, 271-281; Carr, S., et al., Vaccine (1999), 18, 153-159);

[0041] 11. The maltose binding protein has an affinity for amylose (Ahaded, A., et al., Prep. Biochem. Biotechnol. (1999), 29, 163-176).

[0042] Even so, nothing makes it possible to predict whether the operation will actually be successful.

[0043] However, the applicant has just perfected a method which allows human Cdc25B1, Cdc25B2, Cdc25B3 and Cdc25C enzymes to be obtained in an active form and in unlimited quantities. The present invention on one hand facilitates the research and study of the physiological or/and physio-pathological actions of this protein and on the other hand facilitates research for agents modulating these activities.

[0044] The invention makes it possible to obtain a recombinant human Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C protein with the maltose binding protein (MBP) which retains its phosphatase activity without necessitating separation with the MBP part, thus preventing any contamination with the proteases. The high level of the expression rate of the protein after induction allows an excellent purification yield and preparation of the enzyme in unlimited quantities.

[0045] Therefore a subject of the invention is firstly a fusion protein between the maltose binding protein (MBP) and a protein chosen from the Cdc25B1, Cdc25B2, Cdc25B3 and Cdc25C proteins.

[0046] The present invention in particular relates to a protein chosen from the following proteins:

[0047] a fusion protein between human Cdc25B 1 phophatase and the MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 12 (represented further on);

[0048] a fusion protein between human Cdc25B2 phophatase and the MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 13 (represented further on);

[0049] a fusion protein between human Cdc25B3 phophatase and the MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 14 (represented further on); or

[0050] a fusion protein between human Cdc25C phosphatase and the MBP, which is characterized in that it is coded by the sequence SEQ. ID No. 1 (represented further on).

[0051] A subject of the invention is also the DNA coding for the said fusion proteins, as well as the DNA complementary to the DNA coding for the said fusion protein.

[0052] In addition a subject of the invention is the bacterial strain JM 109 transfected:

[0053] by the plasmid with sequence SEQ. ID No. 9 (represented further on), said strain being useful in the preparation of the Cdc25B1 protein;

[0054] by the plasmid with sequence SEQ. ID No. 10 (represented further on), said strain being useful in the preparation of the Cdc25B2 protein;

[0055] by the plasmid with sequence SEQ. ID No. 11 (represented further on), said strain being useful in the preparation of the Cdc25B3 protein; or

[0056] by the plasmid with sequence SEQ. ID No. 5 (represented further on), said strain being useful in the preparation of the Cdc25C protein.

[0057] In addition, the invention relates to a process for the preparation of the said fusion proteins, characterized in that it comprises the following successive stages:

[0058] culture of the bacterial strain JM 109 transfected by the plasmid with sequence SEQ. ID No. 9, the plasmid of sequence SEQ. ID No. 10, the plasmid with sequence SEQ. ID No. 11 or the plasmid with sequence SEQ. ID No. 5, in an LB medium with added ampicillin;

[0059] induction of the fusion protein synthesis by adding isopropylthiogalactoside;

[0060] lysis of the bacteria;

[0061] purification of the fusion protein obtained by chromatography on amylose-agarose resin and recovery of the fractions containing the purified protein.

[0062] Finally the invention relates to a use of said fusion protein in a method for identifying modulators of the Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C protein, characterized in that it comprises the following successive stages:

[0063] addition, of the fusion protein as obtained by the preparation process described previously and a compound presumed to be a modulator of the Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C protein to a solution of 3-O-methylfluorescein phosphate;

[0064] determination of the quantity of 3-O-methylfluorescein produced in relation to the initial quantity of 3-O-methylfluorescein phosphate.

[0065] Determination of the quantity of 3-O-methylfluorescein produced can be carried out, for example, by measuring the optical density of the solution, of the absorbance linked to 3-O-methylfluorescein at the wavelength of 477 nm, or also by fluorometry using excitation at the wavelength of 475 nm and reading at the wavelength of 510 nm.

[0066] Unless they are defined in another way, all the technical and scientific terms used here have the same meaning as that usually understood by a ordinary specialist of the field to which this invention belongs. Similarly, all the publications, patent applications, all the patents and all other references mentioned here are incorporated by way of reference.

EXPERIMENTAL PART

[0067] A/MBP-Cdc25C Fusion Protein:

[0068] 1—Construction of the Expression Vector for MBP-Cdc25C.

[0069] 1.1—Principle of the System Used.

[0070] The system used (New England Biolabs #800-pMAL™ fusion protein and purification system) is based on the production of a fusion protein between the protein of interest, here the human Cdc25C protein, and the bacterial protein MBP (Maltose-Binding Protein) of Escherichia coli. This method allows a one stage purification of the fusion protein due to the affinity of the MBP for maltose.

[0071] 1.2—Origin of the cDNA of Cdc25C

[0072] The DNA coding for human Cdc25C phosphatase corresponds to accession number 4502706.

[0073] Starting from this plasmid, the DNA coding for Cdc25C was amplified by polymerase chain reaction (PCR) using the C-XBAI SENSE and C-XBAI ANTI-SENSE primers (with sequences SEQ. ID. No.s 3 and 4 respectively) and introduced into the vector pCDNA3-HA at the XbaI site in order to produce the vector Cdc25C pcDNA3-HA. The Cdc25C/XbaI insert was, at this stage, sequenced in its entirety.

[0074] The C-XBAI SENSE primer has as its sequence, sequence SEQ. ID. No. 3 represented below:

[0075] 5′-GTTCTAGAAT GTCTAGAA CTCTTC-3′

[0076] The C-XBAI ANTI-SENSE primer has as its sequence, sequence SEQ. ID. No. 4 represented below:

[0077] 5′-GGCTCTGA GTTGCGC CGG-3′

[0078] 1.3—Construction of the Vector pMAL—Hs Cdc25C

[0079] The vector Cdc25C pcDNA3-HA was digested by XbaI, the Cdc25C/XbaI insert (1456 base pairs) was purified and introduced into the vector pMAL™-c2X (New England Biolabs, #800-76) at the XbaI site, in order to produce the vector pMAL-Hs Cdc25C (with sequence SEQ. ID No. 2). This vector allows the production of a fusion protein MBP-Cdc25C of 868 amino acids, from the Ptac bacterial promotor inducible by isopropylthiogalactoside (IPTG).

[0080] The sequence SEQ. ID. No. 2 of the Hs Cdc25C protein with its XBAI restriction ends is the following:

[0081] (the XBAI sites are continuously underlined, the open reading frame (or ORF) of Cdc25C is underlined by a dotted line)

[0082] 1.4—Creation of the JM109/pMAL—Hs Cdc25C Strain

[0083] The vector Cdc25C pMAL—Hs was introduced into the Escherichia coli (E. coli) JM109 (Stratagene #200271) strain. An isolated colony was selected and the production of a protein with a theoretical apparent molecular weight of 97 kDa after culture in the presence of IPTG was observed by analysis of the total bacterial proteins on denaturing polyacrylamide gel and staining with Coomassie blue. The identity of the fusion protein was then confirmed by western blot and immunodetection with an anti-Cdc25C antibody.

[0084] Finally, the plasmidic DNA isolated from this clone was sequenced in the region corresponding to Cdc25C to verify the absence of mutations or modifications of the sequence which could have been generated during the processes of sub-cloning and/or transformation of DNA.

[0085] The following sequence (designated by SEQ. ID. No. 5) was obtained for the JM109/pMAL—Hs Cdc25C strain:

[0086] (the XBAI sites are underlined by a single continuous line, the ORF of Cdc25C is underlined by a dotted line, the maltose binding protein (MBP) sequence is underlined by a double continuous line and the sequence in bold corresponds to the Plasmidic DNA region isolated from the JM 109 strain and then sequenced).

[0087] This clone can be stored at −80° C. in the form of a saturated culture with added glycerol (final concentration 25%) or “stock glycerol”. This strain will be used for all the subsequent production stages.

[0088] 2—Production and Purification of the Recombinant MBP-Cdc25C Protein:

[0089] NB: except where otherwise indicated, all the chemical reagents are supplied by SIGMA-ALDRICH.

[0090] 2.1—Bacterial Culture and Induction of the Expression of the Fusion Protein

[0091] 50 ml of LB medium+ampicillin 100 μg/ml (LB amp.) are inoculated with 100 μl of stock glycerol from the JM109/pMAL-Cdc25C clone and cultured for 14 to 16 hours at 37° C. under stirring (180 to 220 rpm). This pre-culture is then diluted fifty times (20 ml per litre of medium) in an LB amp. medium +2 g/l of glucose and cultured at 37° C./180 rpm in order to reach an optical density at 600 nm of between 0.55 and 0.60. Synthesis of the fusion protein is then induced by adding IPTG (0.3 mM) at 37° C. over 3 hours. The bacteria are collected by centrifugation, washed once in 40 ml of cold PBS per litre of culture, and the bacterial pellet is then frozen in liquid nitrogen and stored at −80° C.

[0092] The induction is analysed immediately by depositing 2.5.10⁷ cells, removed before and after induction, on denaturing polyacrylamide gel and staining of the proteins with Coomassie blue (FIG. 1, lines 1 and 2 respectively).

[0093] 2.2—Lysis and Extraction.

[0094] A bacterial pellet corresponding to 1 litre of induced culture is thawed in ice, re-suspended in 35 ml of lysis buffer (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 μg/ml lysozyme, 1 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF) and incubated for 45 minutes in ice. The bacterial suspension is then sonicated (4 cycles of 1 min in discontinuous mode 50%, alternated with 1 minutes pause), then centrifuged for 35 minutes at 110 000 g. The supernatant or soluble extract is retained for purification of the MBP-Cdc25C protein (FIG. 1, line 3).

[0095] 2.3—Affinity Purification on Amylose-Agarose Resin.

[0096] For a soluble extract corresponding to 1 litre of induced bacterial culture, 2 ml of amylose-agarose resin (New England Biolabs #800-21) is deposited on an HR 5/10 chromatographic column (Pharmacia) and washed with 20 ml (10 volumes) of column buffer (20 mM Tris-HCl pH 7.4; 250 mM NaCl; 1 mM EDTA; 1 mM DTT; 1 μg/ml leupeptin; 2 μg/ml aprotinin). The soluble extract is passed through the affinity column at a flow rate of 0.15 ml/min; the eluate (i.e. the fraction not retained on the amylose-agarose) is collected for analysis (FIG. 1, line 4). The column is washed with 20 ml (10 volumes) of column buffer. A sample of the affinity matrix after passage of the soluble extract can be optionally analysed (FIG. 1, line 5).

[0097] Elution of the protein of the affinity matrix is carried out with a maltose buffer (20 mM Tris-HCl pH 7.4; 250 mM NaCl; 1 mM EDTA; 1 mM DTT; 10 mM maltose). 20 elution fractions of 0.5 ml are collected. For each fraction, the total protein concentration is evaluated by a Bradford type test and the fraction is analysed by depositing on denaturing polyacrylamide gel and staining with Coomassie blue (FIG. 1, line 6). The fractions in which the complete MBP-Cdc25C fusion protein represents at least 90% of the total proteins are collected together to form a batch, the activity of which is then tested. The batches are stored at −80° C.

[0098] Sequencing of the insert comprised between the XbaI sites produces the following result:

[0099] (the XBAI sites are underlined by a single continuous line, the ORF of Cdc25C is underlined by a dotted line and the sequence in italics correspond to the ORF of the maltose binding protein (MBP))

[0100] The sequence of the obtained MBP-Cdc25C fusion protein therefore corresponds to the sequence SEQ. ID No. 1 represented below: ATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA CTGGCGATGG CCCTGACATT ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTCGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACCGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGCGA AAGATCCACG TATTGCCGCC ACCATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAGG ATTTCAGAAT TCGGATCCTC TAGAATGTCT ACGGAACTCT TCTCATCCAC AAGAGAGGAA GGAAGCTCTG GCTCAGGACC CAGTTTTAGG TCTAATCAAA GGAAAATGTT AAACCTGCTC CTGGAGAGAG ACACTTCCTT TACCGTCTGT CCAGATGTCC CTAGAACTCC AGTGGGCAAA TTTCTTGGTG ATTCTGCAAA CCTAAGCATT TTGTCTGGAG GAACCCCAAA ATGTTGCCTC GATCTTTCGA ATCTTAGCAG TGGGGAGATA ACTGCCACTC AGCTTACCAC TTCTGCAGAC CTTGATGAAA CTGGTCACCT GGATTCTTCA GGACTTCAGG AAGTGCATTT AGCTGGGATG AATCATGACC AGCACCTAAT GAAATGTAGC CCAGCACAGC TTCTTTGTAG CACTCCGAAT GGTTTGGACC GTGGCCATAG AAAGAGAGAT GCAATGTGTA GTTCATCTGC AAATAAAGAA AATGACAATG GAAACTTGGT GGACAGTGAA ATGAAATATT TGGGCAGTCC CATTACTACT GTTCCAAAAT TGGATAAAAA TCCAAACCTA CCAGAAGACC AGGCAGAAGA GATTTCAGAT GAATTAATGG AGTTTTCCCT GAAAGATCAA CAAGCAAAGG TGAGCAGAAG TGGCCTATAT CGCTCCCCGT CGATGCCAGA GAACTTGAAC AGGCCAAGAC TGAAGCAGGT GGAAAAATTC AAGGACAACA CAATACCACA TAAAGTTAAA AAAAAGTATT TTTCTGGCCA ACGAAAGCTC AGGAAGGCCT TATGTTTAAA GAAGACAGTC TCTCTGTGTG ACATTACTAT CACTCAGATG CTGGAGGAAG ATTCTAACCA GGGGCACCTG ATTGGTGATT TTTCCAAGGT ATGTGCGCTG CCAACCGTGT CAGGGAAACA CCAAGATCTG AAGTATGTCA ACCCAGAAAC AGTGGCTGCC TTACTGTCGG GGAAGTTCCA GGGTCTGATT GAGAAGTTTT ATGTCATTGA TTGTCGCTAT CCATATGAGT ATCTGGGAGG ACACATCCAG GGAGCCTTAA ACTTATATAG TCAGGAAGAA CTGTTTAACT TCTTTCTGAA GAAGCCCATC GTCCCTTTGG ACACCCAGAA GAGAATAATC ATCGTGTTCC ACTGTGAATT CTCCTCAGAG AGGGGCCCCC GAATGTGCCG CTGTCTGCGT GAAGAGGACA GGTCTCTGAA CCAGTATCCT GCATTGTACT ACCCAGAGCT ATATATCCTT AAAGGCGGCT ACAGAGACTT CTTTCCAGAA TATATGGAAC TGTGTGAACC ACAGAGCTAC TGCCCTATGC ATCATCAGGA CCACAAGACT GAGTTGCTGA GGTGTCGAAG CCAGAGCAAA GTGCAGGAAG GGGAGCGGCA GCTGCGGGAG CAGATTGCCC TTCTGGTGAA GGACATGAGC CCATGA

[0101] 3—Determination of the Activity of the MBP-Cdc25C Fusion Protein:

[0102] The phosphatase activity of the MBP-Cdc25C protein is evaluated by a test of the dephosphorylation of 3-O-methylfluoresceine phosphate (OMFP) with determination of the absorbance at 477 nm (OD 477 nm) of the product of the reaction (OMF).

[0103] The MBP-Cdc25C protein, stored in elution buffer (the same as that described in paragraph 2.3), is diluted to the concentration of 20 nM in the phosphatase reaction buffer (50 mM Tris-HCl pH 8.2; 50 mM NaCl; 1 mM DTT; 20% glycerol), at ambient temperature, in a total reaction volume of 1 ml. The reaction is initiated by the addition of a 0.3 mM solution of OMFP (prepared extemporaneously from a 7.5 mM stock solution in 100% DMSO (Sigma #M2629)) and takes place at 25° C. in a disposable spectrophotometry polystyrene cuvet (Fisher Scientific #A12-103-056). The OD 477 nm is measured after 90 minutes. The reference for the absorbance measurement is constituted by the reaction buffer containing 0.3 mM of OMFP, without MBP-Cdc25C protein, at time t₀ of the reaction. A representative example of the results of such a determination of activity is illustrated by FIG. 2.

[0104] B/MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3 Fusion Proteins:

[0105] 1—Construction of the Expression Vector for MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3

[0106] 1.1—Principle of the System Used.

[0107] The system used (New England Biolabs #800-pMAL™ fusion protein and purification system) is based on the production of a fusion protein between the protein of interest, here the human protein Cdc25B, and the bacterial protein MBP (Maltose-Binding Protein) of Escherichia coli. This method allows a one stage purification of the fusion protein due to the affinity of the MBP for maltose.

[0108] 1.2—Origin of the cDNA of Cdc25B

[0109] The DNA coding for the three splicing variants of human Cdc25B phophatase corresponds to the accession numbers: M81934 and Z68092.

[0110] 1.3—Construction of the Vectors pMAL-Hs Cdc25B

[0111] The differences between the DNA of the 3 Cdc25B variants concern two exons coding for domains of 14 and 42 amino acids located in the regulatory region of the molecule (Baldin et al., Oncogene (1997), 14, 2485-2490) and does not affect its 5′ and 3′ regions. The cloning strategy used for these three variants is therefore identical.

[0112] The cDNA of Cdc25B in the form of NdeI(Klenow)/BamHI inserts were introduced into the pMAL™-C2X vector (New England Biolabs, #800-76) at the EcoR1 (Klenow)/BamH1 site in order to produce the pMAL-Hs Cdc25B1, pMAL-Hs Cdc25B2 and pMAL-Hs Cdc25B3 vectors respectively. These vectors allow the production of the MBP-Cdc25B1 (962 amino acids), MPB-Cdc25B2 (933 amino acids) and MBP-Cdc25B3 (974 amino acids) fusion proteins from the Ptac bacterial promotor inducible by isopropylthiogalactoside (IPTG).

[0113] The sequence SEQ. ID. No. 6 of the Hs Cdc25B1 protein with its NdeI and BamH1 restriction ends is the following: CATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGCCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCTC CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATG CACGACCTCG CCGGGCTCGG CAGCCGCAGC CGCCTGACGC ACCTATCCCT GTCTCGACGG GCATCCGAAT CCTCCCTGTC GTCTGAATCC TCCGAATCTT CTGATGCAGG TCTCTGCATG GATTCCCCCA GCCCTATGGA CCCCCACATG GCGGAGCAGA CGTTTGAACA GGCCATCCAG GCAGCCAGCC GGATCATTCG AAACGAGCAG TTTGCCATCA GACGCTTCCA GTCTATGCCG GTGAGGCTGC TGGGCCACAG CCCCGTGCTT CGGAACATCA CCAACTCCCA GGCGCCCGAC GGCCGGAGGA AGAGCGAGGC GGGCAGTGGA GCTGCCAGCA GCTCTGGGGA AGACAAGGAG AATGATGGAT TTGTCTTCAA GATGCCATGG AAGCCCACAC ATCCCAGCTC CACCCATGCT CTGGCAGAGT GGGCCAGCCG CAGGGAAGCC TTTGCCCAGA GACCCAGCTC GGCCCCCGAC CTGATGTGTC TCAGTCCTGA CCGGAAGATG GAAGTGGAGG AGCTCAGCCC CCTGGCCCTA GGTCGCTTCT CTCTGACCCC TGCAGAGGGG GATACTCAGG AAGATGATGG ATTTGTGGAC ATCCTAGAGA GTGACTTAAA GGATGATGAT GCAGTTCCCC CAGGCATGGA GAGTCTCATT AGTGCCCCAC TGGTCAAGAC CTTGGAAAAG GAAGAGGAAA AGGACCTCGT CATGTACAGC AAGTGCCAGC GGCTCTTCCG CTCTCCGTCC ATGCCCTGCA GCGTGATCCG GCCCATCCTC AAGAGGCTGG AGCGGCCCCA GGACAGGGAC ACGCCCGTGC AGAATAAGCG GAGGCGGAGC GTGACCCCTC CTGAGCAGCA GCAQGAGGCT GAGGAACCTA AAGCCCGCGT CCTCCGCTCA AAATCACTGT GTCACGATGA GATCGAGAAC CTCCTGGACA GTGACCACCG AGAGCTGATT GGAGATTACT CTAAGGCCTT CCTCCTACAG ACAGTAGACG GAAAGCACCA AGACCTCAAG TACATCTCAC CAGAAACGAT GGTGGCCCTA TTGACGGGCA AGTTCAGCAA CATCGTGGAT AAGTTTGTGA TTGTAGACTG CAGATACCCC TATGAATATG AAGGCGGGCA CATCAAGACT GCGGTGAACT TGCCCCTGGA ACGCGACGCC GAGAGCTTCC TACTGAAGAG CCCCATCGCG CCCTGTAGCC TGGACAAGAG AGTCATCCTC ATTTTCCACT GTGAATTCTC ATCTGAGCGT GGGCCCCGCA TGTGCCGTTT CATCAGGGAA CGAGACCGTG CTGTCAACGA CTACCCCAGC CTCTACTACC CTGAGATGTA TATCCTGAAA GGCGGCTACA AGGAGTTCTT CCCTCAGCAC CCGAACTTCT GTGAACCCCA GGACTACCGG CCCATGAACC ACGAGGCCTT CAAGGATGAG CTAAAGACCT TCCGCCTCAA GACTCGCAGC TGGGCTGGGG AGCGGAGCCG GCGGGAGCTC TGTAGCCGGC TGCAGGACCA GTGAGGGGCC TGCGCCAGTC CTGCTACCTC CCTTGCCTTT CGAGGCCTGA AGCCAGCTGC CCTATGGGCC TCCCGGGCTG AGGGCCTCCT GGAGGCCTCA GGTGCTGTCC ATGGGAAAGA TGGTGTGGTG TCCTGCCTGT CTGCCCCAGC CCAGATTCCC CTGTGTCATC CCATCATTTT CCATATCCTG GTGCCCCCCA CCCCTGGAAG AGCCCAGTCT GTTGAGTTAG TTAAGTTGGG TTAATACCAG CTTAAAGGCA GTATTTTGTG TCCTCCAGGA GCTTCTTGTT TCCTTGTTAG GGTTAACCCT TCATCTTCCT GTGTCCTGAA ACGCTCCTTT GTGTGTGTGT CAGCTGAGGA TCC

[0114] (the NdeI and BamH1 sites are continuously underlined)

[0115] The sequence SEQ. ID. No. 7 of the Hs Cdc25B2 protein with its NdeI and BamH1 restriction ends is the following: CATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCTC CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATG CACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGOTAGGGAC CCTGCTCTTC CGCAGCCGCA GCCGCCTGAC GCACCTATCC CTGTCTCGAC GGGCATCCGA ATCCTCCCTG TCGTCTGAAT CCTCCGAATC TTCTGATGCA GGTCTCTGCA TGGATTCCCC CAGCCCTATG GACCCCCACA TGGCGGAGCA GACGTTTGAA CAGGCCATCC AGGCAGCCAG CCGGATCATT CGAAACGAGC AGTTTGCCAT CAGACGCTTC CAGTCTATGC CGGATGGATT TGTCTTCAAG ATGCCATGGA AGCCCACACA TCCCAGCTCC ACCCATGCTC TGGCAGAGTG GGCCAGCCGC AGGGAAGCCT TTGCCCAGAG ACCCAGCTCG GCCCCCGACC TGATGTGTCT CAGTCCTGAC CGGAAGATGG AAGTGGAGGA GCTCAGCCCC CTGGCCCTAG GTCGCTTCTC TCTGACCCCT GCAGAGGGGG ATACTGAGGA AGATGATGGA TTTGTGGACA TCCTAGAGAG TGACTTAAAG GATGATGATG CAGTTCCCCC AGGCATGGAG AGTCTCATTA GTGCCCCACT GGTCAAGACC TTGGAAAAGG AAGAGGAAAA GGACCTCGTC ATGTACAGCA AGTGCCAGCG GCTCTTCCGC TCTCCGTCCA TGCCCTGCAG CGTGATCCGG CCCATCCTCA AGAGGCTGGA GCGGCCCCAG GACAGGGACA CGCCCGTGCA GAATAAGCGG AGGCGGAGCG TGACCCCTCC TGAGGAGCAG CAGGAGGCTG AGGAACCTAA AGCCCGCGTC CTCCGCTCAA AATCACTGTG TCACGATGAG ATCGAGAACC TCCTGGACAG TGACCACCGA GAGCTGATTG GAGATTACTC TAAGGCCTTC CTCCTACAGA CAGTAGACGG AAAGCACCAA GACCTCAAGT ACATCTCACC AGAAACGATG GTGGCCCTAT TGACGGGCAA GTTCAGCAAC ATCGTGGATA AGTTTGTGAT TGTAGACTGC AGATACCCCT ATGAATATGA AGGCGGGCAC ATCAAGACTG CGGTGAACTT GCCCCTGGAA CGCGACGCCG AGAGCTTCCT ACTGAAGAGC CCCATCGCGC CCTGTAGCCT GGACAAGAGA GTCATCCTCA TTTTCCACTG TGAATTCTCA TCTGAGCGTG GGCCCCGCAT GTGCCGTTTC ATCAGGGAAC GAGACCGTGC TGTCAACGAC TACCCCAGCC TCTACTACCC TGAGATGTAT ATCCTGAAAG GCGGCTACAA GGAGTTCTTC CCTCAGCACC CGAACTTCTG TGAACCCCAG GACTACCGGC CCATGAACCA CGAGGCCTTC AAGGATGAGC TAAAGACCTT CCGCCTCAAG ACTCGCAGCT GGGCTGGGGA GCGGAGCCGG CGGGAGCTCT GTAGCCGGCT GCAGGACCAG TGAGGGGCCT GCGCCAGTCC TGCTACCTCC CTTGCCTTTC GAGGCCTGAA GCCAGCTGCC CTATGGGCCT GCCGGGCTGA GGGCCTGCTG GAGGCCTCAG GTGCTGTCCA TGGGAAAGAT GGTGTGGTGT CCTGCCTGTC TGCCCCAGCC CAGATTCCCC TGTGTCATCC CATCATTTTC CATATCCTGG TGCCCCCCAC CCCTGGAAGA GCCCAGTCTG TTGAGTTAGT TAAGTTGGGT TAATACCAGC TTAAAGGCAG TATTTTGTGT CCTCCAGGAG CTTCTTGTTT CCTTGTTAGG GTTAACCCTT CATCTTCCTG TGTCCTGAAA CGCTCCTTTG TGTGTGTGTC AGCTGAGGAT CC

[0116] (the NdeI and BamH1 sites are continuously underlined)

[0117] The sequence SEQ. ID. No. 8 of the Hs Cdc25B3 protein with its NdeI and BamH1 restriction ends is the following: CATATGGAGG TGCCCCAGCC GGAGCCCGCG CCAGGCTCGG CTCTCAGTCC AGCAGGCGTG TGCGGTGGCG CCCAGCGTCC GGGCCACCTC CCGGGCCTCC TGCTGGGATC TCATGGCCTC CTGGGGTCCC CGGTGCGGGC GGCCGCTTCC TCGCCGGTCA CCACCCTCAC CCAGACCATG CACGACCTCG CCGGGCTCGG CAGCGAAACC CCAAAGAGTC AGGTAGCGAC CCTGCTCTTC CGCAGCCGCA GCCGCCTGAC GCACCTATCC CTGTCTCGAC GGGCATCCGA ATCCTCCCTG TCGTCTGAAT CCTCCGAATC TTCTGATGCA GGTCTCTGCA TGGATTCCCC CAGCCCTATG GACCCCCACA TGGCGGAGCA GACGTTTGAA CAGGCCATCC AGGCAGCCAG CCGGATCATT CGAAACGACC AGTTTGCCAT CAGACGCTTC CAGTCTATGC CGGTGAGGCT GCTGGGCCAC AGCCCCGTCC TTCGGAACAT CACCAACTCC CAGGCGCCCG ACGGCCGGAG GAAGAGCGAG GCGGGCAGTG GAGCTGCCAG CAGCTCTGGG GAAGACAAGG AGAATGATGG ATTTGTCTTC AAGATGCCAT GGAAGCCCAC ACATCCCAGC TCCACCCATG CTCTGGCAGA GTGGGCCAGC CGCAGGGAAG CCTTTGCCCA GAGACCCAGC TCGGCCCCCG ACCTGATGTG TCTCAGTCCT GACCGGAAGA TGGAAGTGGA GGAGCTCAGC CCCCTGGCCC TAGGTCGCTT CTCTCTGACC CCTGCAGAGG GGGATACTGA GGAAGATGAT GGATTTGTGG ACATCCTAGA GAGTGACTTA AAGGATGATG ATGCAGTTCC CCCAGGCATG GAGAGTCTCA TTAGTGCCCC ACTGGTCAAG ACCTTGGAAA AGGAAGAGGA AAAGGACCTC GTCATGTACA GCAAGTGCCA GCGGCTCTTC CGCTCTCCGT CCATGCCCTG CAGCGTGATC CGGCCCATCC TCAAGAGGCT GGAGCGGCCC CAGGACAGGG ACACGCCCGT GCAGAATAAG CGGAGGCGGA GCGTGACCCC TCCTGAGGAG CAGCAGGAGG CTGAGGAACC TAAAGCCCGC GTCCTCCGCT CAAAATCACT GTGTCACGAT GAGATCGAGA ACCTCCTGGA CAGTGACCAC CGAGAGCTGA TTGGAGATTA CTCTAAGCCC TTCCTCCTAC AGACAGTAGA CGGAAAGCAC CAAGACCTCA AGTACATCTC ACCAGAAACG ATGGTGGCCC TATTGACGCG CAAGTTCAGC AACATCGTGG ATAAGTTTGT GATTGTAGAC TGCAGATACC CCTATGAATA TGAAGGCGGG CACATCAAGA CTGCGGTGAA CTTGCCCCTG GAACGCGACG CCGAGAGCTT CCTACTGAAG AGCCCCATCG CGCCCTGTAG CCTGGACAAG AGAGTCATCC TCATTTTCCA CTGTGAATTC TCATCTGAGC GTGGGCCCCG CATGTGCCGT TTCATCAGGG AACGAGACCG TGCTGTCAAC GACTACCCCA GCCTCTACTA CCCTGAGATG TATATCCTGA AAGGCGGCTA CAAGGAGTTC TTCCCTCAGC ACCCGAACTT CTGTGAACCC CAGGACTACC GGCCCATGAA CCACGAGGCC TTCAAGGATG AGCTAAAGAC CTTCCGCCTC AAGACTCGCA GCTGGGCTGG GGAGCGGAGC CGGCGGGAGC TCTGTAGCCG GCTGCAGGAC CAGTGAGGGG CCTGCGCCAG TCCTGCTACC TCCCTTGCCT TTCGAGGCCT GAAGCCAGCT GCCCTATGGG CCTGCCGGGC TGAGGGCCTG CTGGAGGCCT CAGGTGCTGT CCATGGGAAA GATGGTGTGC TGTCCTGCCT GTCTGCCCCA GCCCAGATTC CCCTGTGTCA TCCCATCATT TTCCATATCC TGGTGCCCCC CACCCCTGGA AGAGCCCAGT CTGTTGAGTT AGTTAAGTTG GGTTAATACC AGCTTAAAGG CAGTATTTTG TGTCCTCCAG GAGCTTCTTG TTTCCTTGTT AGGGTTAACC CTTCATCTTC CTGTGTCCTG AAACGCTCCT TTGTGTGTGT GTCAGCTGAG GATCC

[0118] (the NdeI and BamH1 sites are continuously underlined)

[0119] 1.4—Creation of the JM109/pMAL-Cdc25B1, B2 and B3 Strains

[0120] The vectors pMAL-Cdc25B1-3 were each introduced into the Escherichia coli JM109 strain (Stratagene #200271). The colonies were selected on the basis of their ability to produce a fusion protein after culture in the presence of IPTG. The identity of the proteins was verified by immunodetection with polyclonal antibodies directed against Cdc25B.

[0121] The plasmidic DNA isolated from these three clones was sequenced in the region corresponding to Cdc25B. The following sequences were obtained (the part with double underlining corresponds to the ORF of MBP and the part underlined by a dotted line to the ORF of Cdc25B1, Cdc25B2 or Cdc25B3): SEQ. ID No. 9 (pMAL-HsCdc25B1):

SEQ. ID No. 10 (pMAL-HsCdc25B2):

SEQ. ID No. 11 (pMAL-HsCdc25B3):

[0122] These clones are kept at −80° C. in the form of saturated culture with added glycerol (final concentration 25%). These strains are then used for all the subsequent production stages.

[0123] 2—Production and Purification of the Recombinant MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3 Proteins:

[0124] The recombinant MBP-Cdc25B1, B2 and B3 proteins are produced in exactly the same way as described for MBP-Cdc25C.

[0125] The sequences SEQ. ID No. 12, SEQ. ID No. 13 and SEQ. ID No. 14 are obtained for the MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3 fusion proteins respectively: SEQ. ID No. 12 (MBP-Cdc25B1):

SEQ. ID No. 13 (MBP-Cdc25B2):

SEQ. ID No. 14 (MBP-Cdc25B3):

[0126] 3—Determination of the Activity of the MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3 Fusion Proteins:

[0127] The activities of the MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3 fusion proteins are measured under the same conditions as those described for the MBP-Cdc25C fusion protein. The results obtained are given in FIG. 3.

LEGEND OF THE FIGURES

[0128]FIG. 1 (FIG. 1) represents the analysis chromatography relative to the induction of expression of the MBP-Cdc25C fusion protein. Lines 1 and 2 of FIG. 1 correspond respectively to the total JM109/pMAL-Cdc25C extract with or without the addition of IPTG. Line 3 corresponds to the soluble extract. Lines 4 and 5 correspond to the non-retained and retained fractions on amylose-agarose respectively. Finally, line 6 of FIG. 1 corresponds to elution fraction No. 12 which contains practically only fusion protein.

[0129]FIG. 2 (FIG. 2) represents the results of measuring the activity of the recombinant MBP-Cdc25C protein (a “+” signifying that menadione had been added to the sample, a “−” signifying that the sample was not treated with menadione).

[0130]FIG. 3 (FIG. 3) represents the results of measuring the activity of the recombinant MBP-Cdc25B1, MBP-Cdc25B2 and MBP-Cdc25B3 proteins. The reaction is carried out with 300 ng of enzyme per trial. MBP at the same concentration is used in the control. The fluorescence values measured allow the calculation of the slopes: 0.0025 Δfluo/sec for the control, 0.0361 Δfluo/sec for Cdc25B1, 0.0350 Δfluo/sec for Cdc25B2 and 0.0372 Δfluo/sec for Cdc25B3.

1 14 1 2607 DNA Artificial Sequence Description of Artificial Sequence Synthetic MBP-Cdc25C fusion nucleic acid sequence 1 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttcggatcct ctagaatgtc tacggaactc 1200 ttctcatcca caagagagga aggaagctct ggctcaggac ccagttttag gtctaatcaa 1260 aggaaaatgt taaacctgct cctggagaga gacacttcct ttaccgtctg tccagatgtc 1320 cctagaactc cagtgggcaa atttcttggt gattctgcaa acctaagcat tttgtctgga 1380 ggaaccccaa aatgttgcct cgatctttcg aatcttagca gtggggagat aactgccact 1440 cagcttacca cttctgcaga ccttgatgaa actggtcacc tggattcttc aggacttcag 1500 gaagtgcatt tagctgggat gaatcatgac cagcacctaa tgaaatgtag cccagcacag 1560 cttctttgta gcactccgaa tggtttggac cgtggccata gaaagagaga tgcaatgtgt 1620 agttcatctg caaataaaga aaatgacaat ggaaacttgg tggacagtga aatgaaatat 1680 ttgggcagtc ccattactac tgttccaaaa ttggataaaa atccaaacct aggagaagac 1740 caggcagaag agatttcaga tgaattaatg gagttttccc tgaaagatca agaagcaaag 1800 gtgagcagaa gtggcctata tcgctccccg tcgatgccag agaacttgaa caggccaaga 1860 ctgaagcagg tggaaaaatt caaggacaac acaataccag ataaagttaa aaaaaagtat 1920 ttttctggcc aaggaaagct caggaagggc ttatgtttaa agaagacagt ctctctgtgt 1980 gacattacta tcactcagat gctggaggaa gattctaacc aggggcacct gattggtgat 2040 ttttccaagg tatgtgcgct gccaaccgtg tcagggaaac accaagatct gaagtatgtc 2100 aacccagaaa cagtggctgc cttactgtcg gggaagttcc agggtctgat tgagaagttt 2160 tatgtcattg attgtcgcta tccatatgag tatctgggag gacacatcca gggagcctta 2220 aacttatata gtcaggaaga actgtttaac ttctttctga agaagcccat cgtccctttg 2280 gacacccaga agagaataat catcgtgttc cactgtgaat tctcctcaga gaggggcccc 2340 cgaatgtgcc gctgtctgcg tgaagaggac aggtctctga accagtatcc tgcattgtac 2400 tacccagagc tatatatcct taaaggcggc tacagagact tctttccaga atatatggaa 2460 ctgtgtgaac cacagagcta ctgccctatg catcatcagg accacaagac tgagttgctg 2520 aggtgtcgaa gccagagcaa agtgcaggaa ggggagcggc agctgcggga gcagattgcc 2580 cttctggtga aggacatgag cccatga 2607 2 1461 DNA Artificial Sequence Description of Artificial Sequence Synthetic Hs Cdc25C nucleic acid sequence with its XbaI restriction ends 2 tctagaatgt ctacggaact cttctcatcc acaagagagg aaggaagctc tggctcagga 60 cccagtttta ggtctaatca aaggaaaatg ttaaacctgc tcctggagag agacacttcc 120 tttaccgtct gtccagatgt ccctagaact ccagtgggca aatttcttgg tgattctgca 180 aacctaagca ttttgtctgg aggaacccca aaatgttgcc tcgatctttc gaatcttagc 240 agtggggaga taactgccac tcagcttacc acttctgcag accttgatga aactggtcac 300 ctggattctt caggacttca ggaagtgcat ttagctggga tgaatcatga ccagcaccta 360 atgaaatgta gcccagcaca gcttctttgt agcactccga atggtttgga ccgtggccat 420 agaaagagag atgcaatgtg tagttcatct gcaaataaag aaaatgacaa tggaaacttg 480 gtggacagtg aaatgaaata tttgggcagt cccattacta ctgttccaaa attggataaa 540 aatccaaacc taggagaaga ccaggcagaa gagatttcag atgaattaat ggagttttcc 600 ctgaaagatc aagaagcaaa ggtgagcaga agtggcctat atcgctcccc gtcgatgcca 660 gagaacttga acaggccaag actgaagcag gtggaaaaat tcaaggacaa cacaatacca 720 gataaagtta aaaaaaagta tttttctggc caaggaaagc tcaggaaggg cttatgttta 780 aagaagacag tctctctgtg tgacattact atcactcaga tgctggagga agattctaac 840 caggggcacc tgattggtga tttttccaag gtatgtgcgc tgccaaccgt gtcagggaaa 900 caccaagatc tgaagtatgt caacccagaa acagtggctg ccttactgtc ggggaagttc 960 cagggtctga ttgagaagtt ttatgtcatt gattgtcgct atccatatga gtatctggga 1020 ggacacatcc agggagcctt aaacttatat agtcaggaag aactgtttaa cttctttctg 1080 aagaagccca tcgtcccttt ggacacccag aagagaataa tcatcgtgtt ccactgtgaa 1140 ttctcctcag agaggggccc ccgaatgtgc cgctgtctgc gtgaagagga caggtctctg 1200 aaccagtatc ctgcattgta ctacccagag ctatatatcc ttaaaggcgg ctacagagac 1260 ttctttccag aatatatgga actgtgtgaa ccacagagct actgccctat gcatcatcag 1320 gaccacaaga ctgagttgct gaggtgtcga agccagagca aagtgcagga aggggagcgg 1380 cagctgcggg agcagattgc ccttctggtg aaggacatga gcccatgata acattccagc 1440 cactggctgc taacatctag a 1461 3 24 DNA Artificial Sequence Description of Artificial Sequence XbaI SENSE primer 3 gttctagaat gtctagaact cttc 24 4 18 DNA Artificial Sequence Description of Artificial Sequence XbaI ANTI- SENSE primer 4 ggctctgagt tgcgccgg 18 5 8101 DNA Artificial Sequence Description of Artificial Sequence Synthetic JM109 / pMAL - Hs Cdc25C strain 5 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 60 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 120 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 180 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 240 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 300 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 360 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 420 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 480 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 540 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 600 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 660 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 720 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 780 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 840 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg 900 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 960 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1020 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1080 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1140 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 1200 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1260 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1320 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1380 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1440 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1500 gcacttcacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg 1560 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 1620 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 1680 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 1740 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 1800 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 1860 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 1920 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 1980 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 2040 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 2100 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 2160 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 2220 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 2280 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 2340 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 2400 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 2460 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg 2520 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 2580 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg 2640 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcagaattc 2700 ggatcctcta gaatgtctac ggaactcttc tcatccacaa gagaggaagg aagctctggc 2760 tcaggaccca gttttaggtc taatcaaagg aaaatgttaa acctgctcct ggagagagac 2820 acttccttta ccgtctgtcc agatgtccct agaactccag tgggcaaatt tcttggtgat 2880 tctgcaaacc taagcatttt gtctggagga accccaaaat gttgcctcga tctttcgaat 2940 cttagcagtg gggagataac tgccactcag cttaccactt ctgcagacct tgatgaaact 3000 ggtcacctgg attcttcagg acttcaggaa gtgcatttag ctgggatgaa tcatgaccag 3060 cacctaatga aatgtagccc agcacagctt ctttgtagca ctccgaatgg tttggaccgt 3120 ggccatagaa agagagatgc aatgtgtagt tcatctgcaa ataaagaaaa tgacaatgga 3180 aacttggtgg acagtgaaat gaaatatttg ggcagtccca ttactactgt tccaaaattg 3240 gataaaaatc caaacctagg agaagaccag gcagaagaga tttcagatga attaatggag 3300 ttttccctga aagatcaaga agcaaaggtg agcagaagtg gcctatatcg ctccccgtcg 3360 atgccagaga acttgaacag gccaagactg aagcaggtgg aaaaattcaa ggacaacaca 3420 ataccagata aagttaaaaa aaagtatttt tctggccaag gaaagctcag gaagggctta 3480 tgtttaaaga agacagtctc tctgtgtgac attactatca ctcagatgct ggaggaagat 3540 tctaaccagg ggcacctgat tggtgatttt tccaaggtat gtgcgctgcc aaccgtgtca 3600 gggaaacacc aagatctgaa gtatgtcaac ccagaaacag tggctgcctt actgtcgggg 3660 aagttccagg gtctgattga gaagttttat gtcattgatt gtcgctatcc atatgagtat 3720 ctgggaggac acatccaggg agccttaaac ttatatagtc aggaagaact gtttaacttc 3780 tttctgaaga agcccatcgt ccctttggac acccagaaga gaataatcat cgtgttccac 3840 tgtgaattct cctcagagag gggcccccga atgtgccgct gtctgcgtga agaggacagg 3900 tctctgaacc agtatcctgc attgtactac ccagagctat atatccttaa aggcggctac 3960 agagacttct ttccagaata tatggaactg tgtgaaccac agagctactg ccctatgcat 4020 catcaggacc acaagactga gttgctgagg tgtcgaagcc agagcaaagt gcaggaaggg 4080 gagcggcagc tgcgggagca gattgccctt ctggtgaagg acatgagccc atgataacat 4140 tccagccact ggctgctaac atctagagtc gacctgcagg caagcttggc actggccgtc 4200 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 4260 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 4320 cagttgcgca gcctgaatgg cgaatggcag cttggctgtt ttggcggatg agataagatt 4380 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct 4440 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt 4500 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat 4560 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 4620 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc 4680 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc 4740 catcctgacg gatggccttt ttgcgtttct acaaactctt tttgtttatt tttctaaata 4800 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4860 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 4920 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 4980 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 5040 agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct gctatgtggc 5100 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5160 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5220 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5280 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5340 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5400 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5460 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 5520 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5580 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5640 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5700 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5760 ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg aagattgtat 5820 aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt 5880 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag 5940 aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga 6000 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg 6060 aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc 6120 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 6180 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc 6240 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtaa aaggatctag 6300 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 6360 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 6420 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 6480 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 6540 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 6600 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 6660 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 6720 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 6780 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 6840 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 6900 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 6960 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 7020 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 7080 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 7140 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat 7200 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 7260 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac 7320 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 7380 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 7440 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat tcacagatgt 7500 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc 7560 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcacttga tgcctccgtg 7620 taagggggaa tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac 7680 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact 7740 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt 7800 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa 7860 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa 7920 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg 7980 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt 8040 cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgaaat 8100 t 8101 6 2063 DNA Artificial Sequence Description of Artificial Sequence Synthetic Hs Cdc25B1 nucleic acid sequence with its NdeI and BamH1 restriction ends 6 catatggagg tgccccagcc ggagcccgcg ccaggctcgg ctctcagtcc agcaggcgtg 60 tgcggtggcg cccagcgtcc gggccacctc ccgggcctcc tgctgggatc tcatggcctc 120 ctggggtccc cggtgcgggc ggccgcttcc tcgccggtca ccaccctcac ccagaccatg 180 cacgacctcg ccgggctcgg cagccgcagc cgcctgacgc acctatccct gtctcgacgg 240 gcatccgaat cctccctgtc gtctgaatcc tccgaatctt ctgatgcagg tctctgcatg 300 gattccccca gccctatgga cccccacatg gcggagcaga cgtttgaaca ggccatccag 360 gcagccagcc ggatcattcg aaacgagcag tttgccatca gacgcttcca gtctatgccg 420 gtgaggctgc tgggccacag ccccgtgctt cggaacatca ccaactccca ggcgcccgac 480 ggccggagga agagcgaggc gggcagtgga gctgccagca gctctgggga agacaaggag 540 aatgatggat ttgtcttcaa gatgccatgg aagcccacac atcccagctc cacccatgct 600 ctggcagagt gggccagccg cagggaagcc tttgcccaga gacccagctc ggcccccgac 660 ctgatgtgtc tcagtcctga ccggaagatg gaagtggagg agctcagccc cctggcccta 720 ggtcgcttct ctctgacccc tgcagagggg gatactgagg aagatgatgg atttgtggac 780 atcctagaga gtgacttaaa ggatgatgat gcagttcccc caggcatgga gagtctcatt 840 agtgccccac tggtcaagac cttggaaaag gaagaggaaa aggacctcgt catgtacagc 900 aagtgccagc ggctcttccg ctctccgtcc atgccctgca gcgtgatccg gcccatcctc 960 aagaggctgg agcggcccca ggacagggac acgcccgtgc agaataagcg gaggcggagc 1020 gtgacccctc ctgaggagca gcaggaggct gaggaaccta aagcccgcgt cctccgctca 1080 aaatcactgt gtcacgatga gatcgagaac ctcctggaca gtgaccaccg agagctgatt 1140 ggagattact ctaaggcctt cctcctacag acagtagacg gaaagcacca agacctcaag 1200 tacatctcac cagaaacgat ggtggcccta ttgacgggca agttcagcaa catcgtggat 1260 aagtttgtga ttgtagactg cagatacccc tatgaatatg aaggcgggca catcaagact 1320 gcggtgaact tgcccctgga acgcgacgcc gagagcttcc tactgaagag ccccatcgcg 1380 ccctgtagcc tggacaagag agtcatcctc attttccact gtgaattctc atctgagcgt 1440 gggccccgca tgtgccgttt catcagggaa cgagaccgtg ctgtcaacga ctaccccagc 1500 ctctactacc ctgagatgta tatcctgaaa ggcggctaca aggagttctt ccctcagcac 1560 ccgaacttct gtgaacccca ggactaccgg cccatgaacc acgaggcctt caaggatgag 1620 ctaaagacct tccgcctcaa gactcgcagc tgggctgggg agcggagccg gcgggagctc 1680 tgtagccggc tgcaggacca gtgaggggcc tgcgccagtc ctgctacctc ccttgccttt 1740 cgaggcctga agccagctgc cctatgggcc tgccgggctg agggcctgct ggaggcctca 1800 ggtgctgtcc atgggaaaga tggtgtggtg tcctgcctgt ctgccccagc ccagattccc 1860 ctgtgtcatc ccatcatttt ccatatcctg gtgcccccca cccctggaag agcccagtct 1920 gttgagttag ttaagttggg ttaataccag cttaaaggca gtattttgtg tcctccagga 1980 gcttcttgtt tccttgttag ggttaaccct tcatcttcct gtgtcctgaa acgctccttt 2040 gtgtgtgtgt cagctgagga tcc 2063 7 1982 DNA Artificial Sequence Description of Artificial Sequence Synthetic Hs Cdc25B2 nucleic acid sequence with its NdeI and BamH1 restriction ends 7 catatggagg tgccccagcc ggagcccgcg ccaggctcgg ctctcagtcc agcaggcgtg 60 tgcggtggcg cccagcgtcc gggccacctc ccgggcctcc tgctgggatc tcatggcctc 120 ctggggtccc cggtgcgggc ggccgcttcc tcgccggtca ccaccctcac ccagaccatg 180 cacgacctcg ccgggctcgg cagcgaaacc ccaaagagtc aggtagggac cctgctcttc 240 cgcagccgca gccgcctgac gcacctatcc ctgtctcgac gggcatccga atcctccctg 300 tcgtctgaat cctccgaatc ttctgatgca ggtctctgca tggattcccc cagccctatg 360 gacccccaca tggcggagca gacgtttgaa caggccatcc aggcagccag ccggatcatt 420 cgaaacgagc agtttgccat cagacgcttc cagtctatgc cggatggatt tgtcttcaag 480 atgccatgga agcccacaca tcccagctcc acccatgctc tggcagagtg ggccagccgc 540 agggaagcct ttgcccagag acccagctcg gcccccgacc tgatgtgtct cagtcctgac 600 cggaagatgg aagtggagga gctcagcccc ctggccctag gtcgcttctc tctgacccct 660 gcagaggggg atactgagga agatgatgga tttgtggaca tcctagagag tgacttaaag 720 gatgatgatg cagttccccc aggcatggag agtctcatta gtgccccact ggtcaagacc 780 ttggaaaagg aagaggaaaa ggacctcgtc atgtacagca agtgccagcg gctcttccgc 840 tctccgtcca tgccctgcag cgtgatccgg cccatcctca agaggctgga gcggccccag 900 gacagggaca cgcccgtgca gaataagcgg aggcggagcg tgacccctcc tgaggagcag 960 caggaggctg aggaacctaa agcccgcgtc ctccgctcaa aatcactgtg tcacgatgag 1020 atcgagaacc tcctggacag tgaccaccga gagctgattg gagattactc taaggccttc 1080 ctcctacaga cagtagacgg aaagcaccaa gacctcaagt acatctcacc agaaacgatg 1140 gtggccctat tgacgggcaa gttcagcaac atcgtggata agtttgtgat tgtagactgc 1200 agatacccct atgaatatga aggcgggcac atcaagactg cggtgaactt gcccctggaa 1260 cgcgacgccg agagcttcct actgaagagc cccatcgcgc cctgtagcct ggacaagaga 1320 gtcatcctca ttttccactg tgaattctca tctgagcgtg ggccccgcat gtgccgtttc 1380 atcagggaac gagaccgtgc tgtcaacgac taccccagcc tctactaccc tgagatgtat 1440 atcctgaaag gcggctacaa ggagttcttc cctcagcacc cgaacttctg tgaaccccag 1500 gactaccggc ccatgaacca cgaggccttc aaggatgagc taaagacctt ccgcctcaag 1560 actcgcagct gggctgggga gcggagccgg cgggagctct gtagccggct gcaggaccag 1620 tgaggggcct gcgccagtcc tgctacctcc cttgcctttc gaggcctgaa gccagctgcc 1680 ctatgggcct gccgggctga gggcctgctg gaggcctcag gtgctgtcca tgggaaagat 1740 ggtgtggtgt cctgcctgtc tgccccagcc cagattcccc tgtgtcatcc catcattttc 1800 catatcctgg tgccccccac ccctggaaga gcccagtctg ttgagttagt taagttgggt 1860 taataccagc ttaaaggcag tattttgtgt cctccaggag cttcttgttt ccttgttagg 1920 gttaaccctt catcttcctg tgtcctgaaa cgctcctttg tgtgtgtgtc agctgaggat 1980 cc 1982 8 2105 DNA Artificial Sequence Description of Artificial Sequence Synthetic Hs Cdc25B3 nucleic acid sequence with its NdeI and BamH1 restriction ends 8 catatggagg tgccccagcc ggagcccgcg ccaggctcgg ctctcagtcc agcaggcgtg 60 tgcggtggcg cccagcgtcc gggccacctc ccgggcctcc tgctgggatc tcatggcctc 120 ctggggtccc cggtgcgggc ggccgcttcc tcgccggtca ccaccctcac ccagaccatg 180 cacgacctcg ccgggctcgg cagcgaaacc ccaaagagtc aggtagggac cctgctcttc 240 cgcagccgca gccgcctgac gcacctatcc ctgtctcgac gggcatccga atcctccctg 300 tcgtctgaat cctccgaatc ttctgatgca ggtctctgca tggattcccc cagccctatg 360 gacccccaca tggcggagca gacgtttgaa caggccatcc aggcagccag ccggatcatt 420 cgaaacgagc agtttgccat cagacgcttc cagtctatgc cggtgaggct gctgggccac 480 agccccgtgc ttcggaacat caccaactcc caggcgcccg acggccggag gaagagcgag 540 gcgggcagtg gagctgccag cagctctggg gaagacaagg agaatgatgg atttgtcttc 600 aagatgccat ggaagcccac acatcccagc tccacccatg ctctggcaga gtgggccagc 660 cgcagggaag cctttgccca gagacccagc tcggcccccg acctgatgtg tctcagtcct 720 gaccggaaga tggaagtgga ggagctcagc cccctggccc taggtcgctt ctctctgacc 780 cctgcagagg gggatactga ggaagatgat ggatttgtgg acatcctaga gagtgactta 840 aaggatgatg atgcagttcc cccaggcatg gagagtctca ttagtgcccc actggtcaag 900 accttggaaa aggaagagga aaaggacctc gtcatgtaca gcaagtgcca gcggctcttc 960 cgctctccgt ccatgccctg cagcgtgatc cggcccatcc tcaagaggct ggagcggccc 1020 caggacaggg acacgcccgt gcagaataag cggaggcgga gcgtgacccc tcctgaggag 1080 cagcaggagg ctgaggaacc taaagcccgc gtcctccgct caaaatcact gtgtcacgat 1140 gagatcgaga acctcctgga cagtgaccac cgagagctga ttggagatta ctctaaggcc 1200 ttcctcctac agacagtaga cggaaagcac caagacctca agtacatctc accagaaacg 1260 atggtggccc tattgacggg caagttcagc aacatcgtgg ataagtttgt gattgtagac 1320 tgcagatacc cctatgaata tgaaggcggg cacatcaaga ctgcggtgaa cttgcccctg 1380 gaacgcgacg ccgagagctt cctactgaag agccccatcg cgccctgtag cctggacaag 1440 agagtcatcc tcattttcca ctgtgaattc tcatctgagc gtgggccccg catgtgccgt 1500 ttcatcaggg aacgagaccg tgctgtcaac gactacccca gcctctacta ccctgagatg 1560 tatatcctga aaggcggcta caaggagttc ttccctcagc acccgaactt ctgtgaaccc 1620 caggactacc ggcccatgaa ccacgaggcc ttcaaggatg agctaaagac cttccgcctc 1680 aagactcgca gctgggctgg ggagcggagc cggcgggagc tctgtagccg gctgcaggac 1740 cagtgagggg cctgcgccag tcctgctacc tcccttgcct ttcgaggcct gaagccagct 1800 gccctatggg cctgccgggc tgagggcctg ctggaggcct caggtgctgt ccatgggaaa 1860 gatggtgtgg tgtcctgcct gtctgcccca gcccagattc ccctgtgtca tcccatcatt 1920 ttccatatcc tggtgccccc cacccctgga agagcccagt ctgttgagtt agttaagttg 1980 ggttaatacc agcttaaagg cagtattttg tgtcctccag gagcttcttg tttccttgtt 2040 agggttaacc cttcatcttc ctgtgtcctg aaacgctcct ttgtgtgtgt gtcagctgag 2100 gatcc 2105 9 3236 DNA Artificial Sequence Description of Artificial Sequence Synthetic JM109 / pMAL - Hs Cdc25B1 strain 9 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 1200 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 1260 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 1320 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagccgc 1380 agccgcctga cgcacctatc cctgtctcga cgggcatccg aatcctccct gtcgtctgaa 1440 tcctccgaat cttctgatgc aggtctctgc atggattccc ccagccctat ggacccccac 1500 atggcggagc agacgtttga acaggccatc caggcagcca gccggatcat tcgaaacgag 1560 cagtttgcca tcagacgctt ccagtctatg ccggtgaggc tgctgggcca cagccccgtg 1620 cttcggaaca tcaccaactc ccaggcgccc gacggccgga ggaagagcga ggcgggcagt 1680 ggagctgcca gcagctctgg ggaagacaag gagaatgatg gatttgtctt caagatgcca 1740 tggaagccca cacatcccag ctccacccat gctctggcag agtgggccag ccgcagggaa 1800 gcctttgccc agagacccag ctcggccccc gacctgatgt gtctcagtcc tgaccggaag 1860 atggaagtgg aggagctcag ccccctggcc ctaggtcgct tctctctgac ccctgcagag 1920 ggggatactg aggaagatga tggatttgtg gacatcctag agagtgactt aaaggatgat 1980 gatgcagttc ccccaggcat ggagagtctc attagtgccc cactggtcaa gaccttggaa 2040 aaggaagagg aaaaggacct cgtcatgtac agcaagtgcc agcggctctt ccgctctccg 2100 tccatgccct gcagcgtgat ccggcccatc ctcaagaggc tggagcggcc ccaggacagg 2160 gacacgcccg tgcagaataa gcggaggcgg agcgtgaccc ctcctgagga gcagcaggag 2220 gctgaggaac ctaaagcccg cgtcctccgc tcaaaatcac tgtgtcacga tgagatcgag 2280 aacctcctgg acagtgacca ccgagagctg attggagatt actctaaggc cttcctccta 2340 cagacagtag acggaaagca ccaagacctc aagtacatct caccagaaac gatggtggcc 2400 ctattgacgg gcaagttcag caacatcgtg gataagtttg tgattgtaga ctgcagatac 2460 ccctatgaat atgaaggcgg gcacatcaag actgcggtga acttgcccct ggaacgcgac 2520 gccgagagct tcctactgaa gagccccatc gcgccctgta gcctggacaa gagagtcatc 2580 ctcattttcc actgtgaatt ctcatctgag cgtgggcccc gcatgtgccg tttcatcagg 2640 gaacgagacc gtgctgtcaa cgactacccc agcctctact accctgagat gtatatcctg 2700 aaaggcggct acaaggagtt cttccctcag cacccgaact tctgtgaacc ccaggactac 2760 cggcccatga accacgaggc cttcaaggat gagctaaaga ccttccgcct caagactcgc 2820 agctgggctg gggagcggag ccggcgggag ctctgtagcc ggctgcagga ccagtgaggg 2880 gcctgcgcca gtcctgctac ctcccttgcc tttcgaggcc tgaagccagc tgccctatgg 2940 gcctgccggg ctgagggcct gctggaggcc tcaggtgctg tccatgggaa agatggtgtg 3000 gtgtcctgcc tgtctgcccc agcccagatt cccctgtgtc atcccatcat tttccatatc 3060 ctggtgcccc ccacccctgg aagagcccag tctgttgagt tagttaagtt gggttaatac 3120 cagcttaaag gcagtatttt gtgtcctcca ggagcttctt gtttccttgt tagggttaac 3180 ccttcatctt cctgtgtcct gaaacgctcc tttgtgtgtg tgtcagctga ggatcc 3236 10 4955 DNA Artificial Sequence Description of Artificial Sequence Synthetic JM109 / pMAL - Hs Cdc25B2 strain 10 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 1200 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 1260 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 1320 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagccgc 1380 agccgcctga cgcacctatc cctgtctcga cgggcatccg aatcctccct gtcgtctgaa 1440 tcctccgaat cttctgatgc aggtctctgc atggattccc ccagccctat ggacccccac 1500 atggcggagc agacgtttga acaggccatc caggcagcca gccggatcat tcgaaacgag 1560 cagtttgcca tcagacgctt ccagtctatg ccggtgaggc tgctgggcca cagccccgtg 1620 cttcggaaca tcaccaactc ccaggcgccc gacggccgga ggaagagcga ggcgggcagt 1680 ggagctgcca gcagctctgg ggaagacaag gagaatgatg gatttgtctt caagatgcca 1740 tggaagccca cacatcccag ctccacccat gctctggcag agtgggccag ccgcagggaa 1800 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 1860 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 1920 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 1980 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 2040 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 2100 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 2160 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 2220 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 2280 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 2340 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 2400 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 2460 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 2520 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 2580 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 2640 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 2700 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 2760 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 2820 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 2880 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 2940 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 3000 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 3060 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 3120 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagcgaa 3180 accccaaaga gtcaggtagg gaccctgctc ttccgcagcc gcagccgcct gacgcaccta 3240 tccctgtctc gacgggcatc cgaatcctcc ctgtcgtctg aatcctccga atcttctgat 3300 gcaggtctct gcatggattc ccccagccct atggaccccc acatggcgga gcagacgttt 3360 gaacaggcca tccaggcagc cagccggatc attcgaaacg agcagtttgc catcagacgc 3420 ttccagtcta tgccggatgg atttgtcttc aagatgccat ggaagcccac acatcccagc 3480 tccacccatg ctctggcaga gtgggccagc cgcagggaag cctttgccca gagacccagc 3540 tcggcccccg acctgatgtg tctcagtcct gaccggaaga tggaagtgga ggagctcagc 3600 cccctggccc taggtcgctt ctctctgacc cctgcagagg gggatactga ggaagatgat 3660 ggatttgtgg acatcctaga gagtgactta aaggatgatg atgcagttcc cccaggcatg 3720 gagagtctca ttagtgcccc actggtcaag accttggaaa aggaagagga aaaggacctc 3780 gtcatgtaca gcaagtgcca gcggctcttc cgctctccgt ccatgccctg cagcgtgatc 3840 cggcccatcc tcaagaggct ggagcggccc caggacaggg acacgcccgt gcagaataag 3900 cggaggcgga gcgtgacccc tcctgaggag cagcaggagg ctgaggaacc taaagcccgc 3960 gtcctccgct caaaatcact gtgtcacgat gagatcgaga acctcctgga cagtgaccac 4020 cgagagctga ttggagatta ctctaaggcc ttcctcctac agacagtaga cggaaagcac 4080 caagacctca agtacatctc accagaaacg atggtggccc tattgacggg caagttcagc 4140 aacatcgtgg ataagtttgt gattgtagac tgcagatacc cctatgaata tgaaggcggg 4200 cacatcaaga ctgcggtgaa cttgcccctg gaacgcgacg ccgagagctt cctactgaag 4260 agccccatcg cgccctgtag cctggacaag agagtcatcc tcattttcca ctgtgaattc 4320 tcatctgagc gtgggccccg catgtgccgt ttcatcaggg aacgagaccg tgctgtcaac 4380 gactacccca gcctctacta ccctgagatg tatatcctga aaggcggcta caaggagttc 4440 ttccctcagc acccgaactt ctgtgaaccc caggactacc ggcccatgaa ccacgaggcc 4500 ttcaaggatg agctaaagac cttccgcctc aagactcgca gctgggctgg ggagcggagc 4560 cggcgggagc tctgtagccg gctgcaggac cagtgagggg cctgcgccag tcctgctacc 4620 tcccttgcct ttcgaggcct gaagccagct gccctatggg cctgccgggc tgagggcctg 4680 ctggaggcct caggtgctgt ccatgggaaa gatggtgtgg tgtcctgcct gtctgcccca 4740 gcccagattc ccctgtgtca tcccatcatt ttccatatcc tggtgccccc cacccctgga 4800 agagcccagt ctgttgagtt agttaagttg ggttaatacc agcttaaagg cagtattttg 4860 tgtcctccag gagcttcttg tttccttgtt agggttaacc cttcatcttc ctgtgtcctg 4920 aaacgctcct ttgtgtgtgt gtcagctgag gatcc 4955 11 3278 DNA Artificial Sequence Description of Artificial Sequence Synthetic JM109 / pMAL - Hs Cdc25B3 strain 11 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 1200 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 1260 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 1320 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagcgaa 1380 accccaaaga gtcaggtagg gaccctgctc ttccgcagcc gcagccgcct gacgcaccta 1440 tccctgtctc gacgggcatc cgaatcctcc ctgtcgtctg aatcctccga atcttctgat 1500 gcaggtctct gcatggattc ccccagccct atggaccccc acatggcgga gcagacgttt 1560 gaacaggcca tccaggcagc cagccggatc attcgaaacg agcagtttgc catcagacgc 1620 ttccagtcta tgccggtgag gctgctgggc cacagccccg tgcttcggaa catcaccaac 1680 tcccaggcgc ccgacggccg gaggaagagc gaggcgggca gtggagctgc cagcagctct 1740 ggggaagaca aggagaatga tggatttgtc ttcaagatgc catggaagcc cacacatccc 1800 agctccaccc atgctctggc agagtgggcc agccgcaggg aagcctttgc ccagagaccc 1860 agctcggccc ccgacctgat gtgtctcagt cctgaccgga agatggaagt ggaggagctc 1920 agccccctgg ccctaggtcg cttctctctg acccctgcag agggggatac tgaggaagat 1980 gatggatttg tggacatcct agagagtgac ttaaaggatg atgatgcagt tcccccaggc 2040 atggagagtc tcattagtgc cccactggtc aagaccttgg aaaaggaaga ggaaaaggac 2100 ctcgtcatgt acagcaagtg ccagcggctc ttccgctctc cgtccatgcc ctgcagcgtg 2160 atccggccca tcctcaagag gctggagcgg ccccaggaca gggacacgcc cgtgcagaat 2220 aagcggaggc ggagcgtgac ccctcctgag gagcagcagg aggctgagga acctaaagcc 2280 cgcgtcctcc gctcaaaatc actgtgtcac gatgagatcg agaacctcct ggacagtgac 2340 caccgagagc tgattggaga ttactctaag gccttcctcc tacagacagt agacggaaag 2400 caccaagacc tcaagtacat ctcaccagaa acgatggtgg ccctattgac gggcaagttc 2460 agcaacatcg tggataagtt tgtgattgta gactgcagat acccctatga atatgaaggc 2520 gggcacatca agactgcggt gaacttgccc ctggaacgcg acgccgagag cttcctactg 2580 aagagcccca tcgcgccctg tagcctggac aagagagtca tcctcatttt ccactgtgaa 2640 ttctcatctg agcgtgggcc ccgcatgtgc cgtttcatca gggaacgaga ccgtgctgtc 2700 aacgactacc ccagcctcta ctaccctgag atgtatatcc tgaaaggcgg ctacaaggag 2760 ttcttccctc agcacccgaa cttctgtgaa ccccaggact accggcccat gaaccacgag 2820 gccttcaagg atgagctaaa gaccttccgc ctcaagactc gcagctgggc tggggagcgg 2880 agccggcggg agctctgtag ccggctgcag gaccagtgag gggcctgcgc cagtcctgct 2940 acctcccttg cctttcgagg cctgaagcca gctgccctat gggcctgccg ggctgagggc 3000 ctgctggagg cctcaggtgc tgtccatggg aaagatggtg tggtgtcctg cctgtctgcc 3060 ccagcccaga ttcccctgtg tcatcccatc attttccata tcctggtgcc ccccacccct 3120 ggaagagccc agtctgttga gttagttaag ttgggttaat accagcttaa aggcagtatt 3180 ttgtgtcctc caggagcttc ttgtttcctt gttagggtta acccttcatc ttcctgtgtc 3240 ctgaaacgct cctttgtgtg tgtgtcagct gaggatcc 3278 12 2877 DNA Artificial Sequence Description of Artificial Sequence Synthetic MBP-Cdc25B1 fusion nucleic acid sequence 12 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 1200 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 1260 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 1320 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagccgc 1380 agccgcctga cgcacctatc cctgtctcga cgggcatccg aatcctccct gtcgtctgaa 1440 tcctccgaat cttctgatgc aggtctctgc atggattccc ccagccctat ggacccccac 1500 atggcggagc agacgtttga acaggccatc caggcagcca gccggatcat tcgaaacgag 1560 cagtttgcca tcagacgctt ccagtctatg ccggtgaggc tgctgggcca cagccccgtg 1620 cttcggaaca tcaccaactc ccaggcgccc gacggccgga ggaagagcga ggcgggcagt 1680 ggagctgcca gcagctctgg ggaagacaag gagaatgatg gatttgtctt caagatgcca 1740 tggaagccca cacatcccag ctccacccat gctctggcag agtgggccag ccgcagggaa 1800 gcctttgccc agagacccag ctcggccccc gacctgatgt gtctcagtcc tgaccggaag 1860 atggaagtgg aggagctcag ccccctggcc ctaggtcgct tctctctgac ccctgcagag 1920 ggggatactg aggaagatga tggatttgtg gacatcctag agagtgactt aaaggatgat 1980 gatgcagttc ccccaggcat ggagagtctc attagtgccc cactggtcaa gaccttggaa 2040 aaggaagagg aaaaggacct cgtcatgtac agcaagtgcc agcggctctt ccgctctccg 2100 tccatgccct gcagcgtgat ccggcccatc ctcaagaggc tggagcggcc ccaggacagg 2160 gacacgcccg tgcagaataa gcggaggcgg agcgtgaccc ctcctgagga gcagcaggag 2220 gctgaggaac ctaaagcccg cgtcctccgc tcaaaatcac tgtgtcacga tgagatcgag 2280 aacctcctgg acagtgacca ccgagagctg attggagatt actctaaggc cttcctccta 2340 cagacagtag acggaaagca ccaagacctc aagtacatct caccagaaac gatggtggcc 2400 ctattgacgg gcaagttcag caacatcgtg gataagtttg tgattgtaga ctgcagatac 2460 ccctatgaat atgaaggcgg gcacatcaag actgcggtga acttgcccct ggaacgcgac 2520 gccgagagct tcctactgaa gagccccatc gcgccctgta gcctggacaa gagagtcatc 2580 ctcattttcc actgtgaatt ctcatctgag cgtgggcccc gcatgtgccg tttcatcagg 2640 gaacgagacc gtgctgtcaa cgactacccc agcctctact accctgagat gtatatcctg 2700 aaaggcggct acaaggagtt cttccctcag cacccgaact tctgtgaacc ccaggactac 2760 cggcccatga accacgaggc cttcaaggat gagctaaaga ccttccgcct caagactcgc 2820 agctgggctg gggagcggag ccggcgggag ctctgtagcc ggctgcagga ccagtga 2877 13 2796 DNA Artificial Sequence Description of Artificial Sequence Synthetic MBP-Cdc25B2 fusion nucleic acid sequence 13 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 1200 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 1260 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 1320 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagcgaa 1380 accccaaaga gtcaggtagg gaccctgctc ttccgcagcc gcagccgcct gacgcaccta 1440 tccctgtctc gacgggcatc cgaatcctcc ctgtcgtctg aatcctccga atcttctgat 1500 gcaggtctct gcatggattc ccccagccct atggaccccc acatggcgga gcagacgttt 1560 gaacaggcca tccaggcagc cagccggatc attcgaaacg agcagtttgc catcagacgc 1620 ttccagtcta tgccggatgg atttgtcttc aagatgccat ggaagcccac acatcccagc 1680 tccacccatg ctctggcaga gtgggccagc cgcagggaag cctttgccca gagacccagc 1740 tcggcccccg acctgatgtg tctcagtcct gaccggaaga tggaagtgga ggagctcagc 1800 cccctggccc taggtcgctt ctctctgacc cctgcagagg gggatactga ggaagatgat 1860 ggatttgtgg acatcctaga gagtgactta aaggatgatg atgcagttcc cccaggcatg 1920 gagagtctca ttagtgcccc actggtcaag accttggaaa aggaagagga aaaggacctc 1980 gtcatgtaca gcaagtgcca gcggctcttc cgctctccgt ccatgccctg cagcgtgatc 2040 cggcccatcc tcaagaggct ggagcggccc caggacaggg acacgcccgt gcagaataag 2100 cggaggcgga gcgtgacccc tcctgaggag cagcaggagg ctgaggaacc taaagcccgc 2160 gtcctccgct caaaatcact gtgtcacgat gagatcgaga acctcctgga cagtgaccac 2220 cgagagctga ttggagatta ctctaaggcc ttcctcctac agacagtaga cggaaagcac 2280 caagacctca agtacatctc accagaaacg atggtggccc tattgacggg caagttcagc 2340 aacatcgtgg ataagtttgt gattgtagac tgcagatacc cctatgaata tgaaggcggg 2400 cacatcaaga ctgcggtgaa cttgcccctg gaacgcgacg ccgagagctt cctactgaag 2460 agccccatcg cgccctgtag cctggacaag agagtcatcc tcattttcca ctgtgaattc 2520 tcatctgagc gtgggccccg catgtgccgt ttcatcaggg aacgagaccg tgctgtcaac 2580 gactacccca gcctctacta ccctgagatg tatatcctga aaggcggcta caaggagttc 2640 ttccctcagc acccgaactt ctgtgaaccc caggactacc ggcccatgaa ccacgaggcc 2700 ttcaaggatg agctaaagac cttccgcctc aagactcgca gctgggctgg ggagcggagc 2760 cggcgggagc tctgtagccg gctgcaggac cagtga 2796 14 2919 DNA Artificial Sequence Description of Artificial Sequence Synthetic MBP-Cdc25B3 fusion nucleic acid sequence 14 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttccatatgg aggtgcccca gccggagccc 1200 gcgccaggct cggctctcag tccagcaggc gtgtgcggtg gcgcccagcg tccgggccac 1260 ctcccgggcc tcctgctggg atctcatggc ctcctggggt ccccggtgcg ggcggccgct 1320 tcctcgccgg tcaccaccct cacccagacc atgcacgacc tcgccgggct cggcagcgaa 1380 accccaaaga gtcaggtagg gaccctgctc ttccgcagcc gcagccgcct gacgcaccta 1440 tccctgtctc gacgggcatc cgaatcctcc ctgtcgtctg aatcctccga atcttctgat 1500 gcaggtctct gcatggattc ccccagccct atggaccccc acatggcgga gcagacgttt 1560 gaacaggcca tccaggcagc cagccggatc attcgaaacg agcagtttgc catcagacgc 1620 ttccagtcta tgccggtgag gctgctgggc cacagccccg tgcttcggaa catcaccaac 1680 tcccaggcgc ccgacggccg gaggaagagc gaggcgggca gtggagctgc cagcagctct 1740 ggggaagaca aggagaatga tggatttgtc ttcaagatgc catggaagcc cacacatccc 1800 agctccaccc atgctctggc agagtgggcc agccgcaggg aagcctttgc ccagagaccc 1860 agctcggccc ccgacctgat gtgtctcagt cctgaccgga agatggaagt ggaggagctc 1920 agccccctgg ccctaggtcg cttctctctg acccctgcag agggggatac tgaggaagat 1980 gatggatttg tggacatcct agagagtgac ttaaaggatg atgatgcagt tcccccaggc 2040 atggagagtc tcattagtgc cccactggtc aagaccttgg aaaaggaaga ggaaaaggac 2100 ctcgtcatgt acagcaagtg ccagcggctc ttccgctctc cgtccatgcc ctgcagcgtg 2160 atccggccca tcctcaagag gctggagcgg ccccaggaca gggacacgcc cgtgcagaat 2220 aagcggaggc ggagcgtgac ccctcctgag gagcagcagg aggctgagga acctaaagcc 2280 cgcgtcctcc gctcaaaatc actgtgtcac gatgagatcg agaacctcct ggacagtgac 2340 caccgagagc tgattggaga ttactctaag gccttcctcc tacagacagt agacggaaag 2400 caccaagacc tcaagtacat ctcaccagaa acgatggtgg ccctattgac gggcaagttc 2460 agcaacatcg tggataagtt tgtgattgta gactgcagat acccctatga atatgaaggc 2520 gggcacatca agactgcggt gaacttgccc ctggaacgcg acgccgagag cttcctactg 2580 aagagcccca tcgcgccctg tagcctggac aagagagtca tcctcatttt ccactgtgaa 2640 ttctcatctg agcgtgggcc ccgcatgtgc cgtttcatca gggaacgaga ccgtgctgtc 2700 aacgactacc ccagcctcta ctaccctgag atgtatatcc tgaaaggcgg ctacaaggag 2760 ttcttccctc agcacccgaa cttctgtgaa ccccaggact accggcccat gaaccacgag 2820 gccttcaagg atgagctaaa gaccttccgc ctcaagactc gcagctgggc tggggagcgg 2880 agccggcggg agctctgtag ccggctgcag gaccagtga 2919 

1. Protein characterized in that it is a fusion protein between the maltose binding protein (MBP) and a protein chosen from the Cdc25B1, Cdc25B2, Cdc25B3 and Cdc25C proteins.
 2. Protein according to claim 1, characterized in that it is chosen from the following proteins: a fusion protein between human Cdc25B1 phophatase and the MBP, which is coded by the sequence SEQ. ID No. 12; a fusion protein between human Cdc25B2 phophatase and the MBP, which is coded by the sequence SEQ. ID No. 13; a fusion protein between human Cdc25B3 phophatase and the MBP, which is coded by the sequence SEQ. ID No. 14; and a fusion protein between human Cdc25C phosphatase and the MBP, which is characterized in that it is coded by the sequence SEQ. ID No.
 1. 3. Protein according to claim 2, characterized in that it is coded by the sequence SEQ. ID No.
 1. 4. DNA characterized in that it codes for a protein according to one of claims 1 to
 3. 5. Complementary DNA to the DNA according to claim
 4. 6. Bacterial strain characterized in that it is a JM 109 bacterial strain transfected by a plasmid chosen from the plasmid of sequence SEQ. ID No. 9, the plasmid of sequence SEQ. ID No. 10, the plasmid of sequence SEQ. ID No. 11 and the plasmid of sequence SEQ. ID No.
 5. 7. Process for the preparation of a protein according to claim 1, characterized in that it comprises the following successive stages: culture of the bacterial strain JM 109 transfected by the plasmid of sequence SEQ. ID No. 9, the plasmid of sequence SEQ. ID No. 10, the plasmid of sequence SEQ. ID No. 11 or the plasmid of sequence SEQ. ID No. 5, in an LB medium with added ampicillin; induction of the fusion protein synthesis by adding isopropylthiogalactoside; lysis of the bacteria; purification of the fusion protein obtained by chromatography on amylose-agarose resin and recovery of the fractions containing the purified protein.
 8. Use of a protein according to claim 1 in a method for identifying modulators of the Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C protein, characterized in that said method comprises the following successive stages: addition, of the fusion protein such as obtained by a process for the preparation according to claim 7 and of a compound presumed be a modulator of the Cdc25B1, Cdc25B2, Cdc25B3 or Cdc25C protein to a solution of 3-O-methylfluorescein phosphate; determination of the quantity of 3-O-methylfluorescein produced in relation to the initial quantity of 3-O-methylfluorescein phosphate.
 9. Use according to claim 8, characterized in that the determination of the quantity of 3-O-methylfluorescein produced in relation to the initial quantity of 3-O-methylfluorescein phosphate is carried out by measuring the absorbance linked to 3-O-methylfluoresceine at the wavelength of 477 nm.
 10. Use according to claim 8, characterized in that the determination of the quantity of 3-O-methylfluoresceine produced in relation to the initial quantity of 3-O-methylfluoresceine phosphate is carried out by fluorometry using excitation at the wavelength of 475 nm and reading at the wavelength of 510 nm. 